The effect of lipids on bongkrekic (Bongkrek) acid toxin production by Burkholderia cocovenenans in coconut media

Abstract

Tempe bongkrek is an Indonesian food made by fermentation of coconut presscake or coconut milk residue Rhizopus oligosporus. Consumption of tempe bongkrek is associated with a food-borne human intoxication and significant numbers of deaths annually. The bacterium Burkholderia cocovenenans, which is the causative organism, produces two toxins, toxoflavin and bongkrekic acid (also commonly referred to as bongkrek acid). The reasons why these poisonings occur only in a very limited number of foods and only in isolated regions of the world are unclear. Our preliminary experiments in defined media and coconut investigated several compositional and environmental factors and suggested that lipid type and/or concentration were important. The effect of lipid concentration and fatty acid type on the production of bongkrekic acid by B. cocovenenans was examined by adding different amounts of coconut fat or individual free fatty acids to defatted and sterilized Rich Coconut Media (dRCM). The dRCM with added lipid was inoculated with B. cocovenenans, incubated at 30°C for 5 days and the amount of bongkrekic acid formed quantified by HPLC. Coconut fat concentrations of 10% (dry basis) or less did not result in detectable amounts of bongkrekic acid even though the B. cocovenenans grew to high levels. Forty and 50% coconut fat resulted in as much as 1.4mg/g bonkrekic acid (dry weight) at the same level of growth. Of eight saturated fatty acids tested, only lauric (12:0), myristic (14:0), and palmitic (16:0)acids stimulated the production of detectable amounts of toxin. When four 18-carbon free fatty acids with different degrees of saturation were compared, significant amounts of bongkrekic acid (2.62mg/g dry weight) were produced only with oleic acid (18:1). These data indicate that the concentration and type of lipid in the substrate is critical for bongkrekic acid formation. This may explain why bongkrekic acid intoxication is limited to certain foods. Outbreaks associated with foods containing less than 20% fat may be a result of toxoflavin formation and not bongkrekic acid formation.